Histones, the major proteins of eukaryotic chromosomes, undergo reversible acetylation at certain lysine residues, but this acetylation is drastically reduced during normal mitosis and in metaphase-arrested cells. Because both acetylase and deacetylase activities are involved, three hypotheses could explain this phenomenon: (1) acetylase activity is decreased; (2) deacetylase activity is increased; or (3) acetylation sites are masked at mitosis. The primary goal of this project is to test these three hypotheses and thus determine the general mechanism involved. This problem is interesting and important because histone acetylation correlates with gene activity, which is switched off at mitosis. This work might therefore give clues to mechanisms of transcriptional regulation. The long term aim, to understand the function of this and other biochemical changes which occur in eukaryotic chromosomes during the cell cycle, has obvious implications for the understanding of cancer and genetic disease. Preliminary work suggests that possibility is most likely. It will be tested by comparing the acetylation of interphase and mitotic chromatin in vitro using histone acetylase and/or selective chemical acetylating agents. If differences are observed, they will be explored further; are they dependent on intact higher order structure, histone H1 phosphorylation, or the presence of non- histone proteins? Hypotheses one and two will be tested by comparing the amounts of endogenous acetylase and deacetylase activities, respectively, in extracts of interphase and metaphase-arrested HeLa cells.Hypothesis two will also be tested in vivo. A further goal of the project is to systematically explore the effects of Triton X-100 and urea on Hepes/histidine gel electrophoresis, a new method for rapid analysis of histone modifications, in order to find conditions to resolve each histone and its modified forms. Finally, several in vivo experiments will be carried out to test (a) whether H1 phosphorylation is the cause of reduced histone acetylation at mitosis; (b) whether histone deacetylation is prerequisite to the shut-off of transcription; and (c) whether chromosome decondensation at the end of mitosis depends in part on resumption of transcription.